Functional Characterization of Intracellular and Secreted Forms of a Truncated Hepatitis C Virus E2 Glycoprotein

Flint, Mike; Dubuisson, Jean; Maidens, Catherine; Harrop, Richard; Guile, Geoffrey R.; Borrow, Persephone; McKeating, Jane A.

doi:10.1128/jvi.74.2.702-709.2000

Cited by 106 publications

(104 citation statements)

References 34 publications

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“…The anti-E2 monoclonal antibodies (MAbs) AP33, ALP98, H48, H35, and H53 and a rabbit polyclonal antiserum R646 have been described previously (5,10,15,38). MAbs were purified from hybridoma supernatants using a HiTrap protein G column according to the manufacturer's protocol (GE Healthcare).…”

Section: Methodsmentioning

confidence: 99%

Identification of Conserved Residues in the E2 Envelope Glycoprotein of the Hepatitis C Virus That Are Critical for CD81 Binding

Owsianka

Timms

Tarr

et al. 2006

J Virol

228

268

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Hepatitis C virus (HCV) cell entry involves interaction between the viral envelope glycoprotein E2 and the cell surface receptor CD81. Knowledge of conserved E2 determinants important for successful binding will facilitate development of entry inhibitors designed to block this interaction. Previous studies have assigned the CD81 binding function to a number of discontinuous regions of E2. To better define specific residues involved in receptor binding, a panel of mutants of HCV envelope proteins was generated, where conserved residues within putative CD81 binding regions were sequentially mutated to alanine. Mutant proteins were tested for binding to a panel of monoclonal antibodies and CD81 and for their ability to form noncovalent heterodimers and confer infectivity in the retroviral pseudoparticle (HCVpp) assay. Detection by conformation-sensitive monoclonal antibodies indicated that the mutant proteins were correctly folded. Mutant proteins fell into three groups: those that bound CD81 and conferred HCVpp infectivity, those that abrogated both CD81 binding and HCVpp infectivity, and a final group containing mutants that were able to bind CD81 but were noninfectious in the HCVpp assay. Specific amino acids conserved across all genotypes that were critical for CD81 binding were W420, Y527, W529, G530, and D535. These data significantly increase our understanding of the CD81 receptor-E2 binding process.Hepatitis C virus (HCV) is the sole member of the Hepacivirus genus within the family Flaviviridae. It is a major cause of community-acquired and posttransfusion hepatitis. More than 170 million people worldwide are seropositive for HCV, and only 20% of those infected are able to clear the virus. In the remaining 80% of individuals, the virus persists and can

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Section: Methodsmentioning

confidence: 99%

Identification of Conserved Residues in the E2 Envelope Glycoprotein of the Hepatitis C Virus That Are Critical for CD81 Binding

Owsianka

Timms

Tarr

et al. 2006

J Virol

228

268

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“…Genotype 1a soluble E2 (sE2) containing residues 384 to 661 was generated and its binding to cells was detected with rat anti-E2 monoclonal antibodies 9/75 and 7/59 as previously described. 12,34 HCVpp Generation and Infection. HCVpp were generated as previously described.…”

Section: Methodsmentioning

confidence: 99%

Oxidized low-density lipoprotein inhibits hepatitis C virus cell entry in human hepatoma cells

et al. 2006

Self Cite

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“…The higher molecular weight displayed by the extracellular protein fraction is indicative of a different extent of glycosylation and confirms that the protein is actively secreted and not simply released by lysed cells. 55 In both cases, endoglycosydase treatment led to an increase of the mobility as expected from the amino acid composition (Fig. 4A).…”

Section: Dna Immunization With Full-length or C-terminally Truncatedmentioning

confidence: 80%

“…This is likely caused by the presence of modified glycans, which has been shown to modulate E2 binding on CD81. 55 …”

Section: Dna Immunization With Full-length or C-terminally Truncatedmentioning

confidence: 99%

Mimotopes of the hepatitis C virus hypervariable region 1, but not the natural sequences, induce cross-reactive antibody response by genetic immunization

Zucchelli¹,

Roccasecca²,

Meola³

et al. 2001

Hepatology

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The hypervariable region 1 (HVR1) of the putative envelope protein E2 of hepatitis C virus (HCV) contains a principal neutralization epitope, and anti-HVR1 antibodies have been shown to possess protective activity in ex vivo neutralization experiments. However, the high rate of variability of this antigenic fragment may play a major role in the mechanism of escape from host immune response and might represent a major obstacle to developing an HCV vaccine. Thus, even if direct experimental evidence of the neutralizing potential of anti-HVR1 antibodies by active immunization is still missing, the generation of a vaccine candidate with a cross-reactive potential would be highly desirable. Hepatitis C virus (HCV) is the major etiologic agent of blood transfusion-associated and sporadic non-A non-B hepatitis worldwide, with an estimated prevalence of 0.4% to 2% in the blood donor population. 1 Despite a wide array of humoral and cell-mediated host immune responses, HCV infection leads to chronic disease in about 70% of cases, among which a significant proportion eventually develops cirrhosis and hepatocellular carcinoma. 2 Interferon treatment (also in combination with ribavirin) is the only antiviral therapy available at the moment, but it is effective only in 20% to 40% of patients, 3-5 thus making the development of an HCV vaccine a high priority target.Vaccination of chimpanzees (the only other species susceptible to HCV infection) using recombinant forms of the putative envelope proteins E1 and E2 has been shown to induce protective immunity against low-dose challenge with the homologous virus, whose efficacy seems to correlate with the titer of anti-E2 antibodies. [6][7][8] More recently, HCV was shown to bind to its putative receptor component CD81 via E2. 9 These data point toward the E2 protein as a likely candidate for vaccine development. However, a major concern still remains as to whether the anti-E2 response elicited by one recombinant protein would be effective against heterologous viral inocula. 10 In fact, HCV displays a high rate of mutation during replication and exists in the bloodstream of infected patients as a quasispecies, [11][12][13][14][15] which fluctuates during the course of the disease mainly as a result of immune pressure. [15][16][17][18][19][20][21][22][23] The major target of this immune response is the 27 amino acid-long N-terminal segment of the E2 glycoprotein. This protein fragment is the most variable region of the whole HCV polyprotein 11 and contains a principal neutralization determinant, 24 but anti-hypervariable region 1 (HVR1) antibodies specific for one variant display only a limited ability to block different viral variants. 24 In this scenario, the most difficult task in developing an HCV vaccine would be to find a solution to the issue of viral variability.We have recently described a novel approach to this problem by selecting highly cross-reacting "synthetic variants" of the HCV HVR1 from a vast repertoire of HVR1 surrogates as fusion to the major coat protein of ba...

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Functional Characterization of Intracellular and Secreted Forms of a Truncated Hepatitis C Virus E2 Glycoprotein

Cited by 106 publications

References 34 publications

Identification of Conserved Residues in the E2 Envelope Glycoprotein of the Hepatitis C Virus That Are Critical for CD81 Binding

Identification of Conserved Residues in the E2 Envelope Glycoprotein of the Hepatitis C Virus That Are Critical for CD81 Binding

Oxidized low-density lipoprotein inhibits hepatitis C virus cell entry in human hepatoma cells

Mimotopes of the hepatitis C virus hypervariable region 1, but not the natural sequences, induce cross-reactive antibody response by genetic immunization

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